DEGRADATION OF CHLOROAROMATICS - PURIFICATION AND CHARACTERIZATION OFA NOVEL TYPE OF CHLOROCATECHOL 2,3-DIOXYGENASE OF PSEUDOMONAS-PUTIDA GJ31

A purification procedure for a new kind of extradiol dioxygenase, term ed chlorocatechol 2,3-dioxygenase, that converts 3-chlorocatechol prod uctively was developed. Structural and kinetic properties of the enzym e, which is part of the degradative pathway used for growth of Pseudom onas putida GJ31 with chlorobenzene, were investigated. The enzyme has a subunit molecular mass of 33.4 kDa by sodium dodecyl sulfate-polyac rylamide gel electrophoresis. Estimation of the native M-r value under nondenaturating conditions by gel filtration gave a molecular mass of 135 +/- 10 kDa, indicating a homotetrameric enzyme structure (4 x 33. 4 kDa). The pi of the enzyme was estimated to be 7.1 +/- 0.1. The N-te rminal amino acid sequence (43 residues) of the enzyme was determined and exhibits 70 to 42% identity with other extradiol dioxygenases. Fe( II) seems to be a cofactor of the enzyme, as it is for other catechol 2,3-dioxygenases, In contrast to other extradiol dioxygenases, the enz yme exhibited great sensitivity to temperatures above 40 degrees C. Th e reactivity of this enzyme toward various substituted catechols, espe cially 3-chlorocatechol, was different from that observed for other ca techol 2,3-dioxygenases. Stoichiometric displacement of chloride occur red from 3-chlorocatechol, leading to the production of 2-hydroxymucon ate.